Project Summary DNA methylation is an epigenetic mark found in most eukaryotes. At transposons and promoters, DNA methylation primarily acts as a repressive mark. However, DNA methylation is also commonly found over gene bodies, a phenomenon called gene body methylation (GBM). GBM is not generally associated with repression of marked genes; instead, GBM genes tend to be moderately expressed, longer, and more functionally important than non-GBM genes. GBM is also highly conserved throughout the plant and animal kingdoms. The widespread conservation of GBM in spite of the mutagenic properties of DNA methylation suggests that this type of methylation is functionally important. Loss of GBM is also a hallmark of cancer, and may contribute to the aberrant phenotypes seen in cancerous cells. Yet despite these observations, the function of GBM remains a fundamental open question in epigenetics. Improving our understanding of the function of GBM will not only advance our understanding of epigenetic regulation, but may also provide valuable insights that could be used for clinical applications in cancer treatment. There are currently three major hypothesized functions for GBM. The first hypothesis, originally proposed based on the observation that DNA methylation is higher in exons than in introns, is that GBM modulates splicing. Another hypothesis is that GBM is involved in regulating gene expression levels. Finally, a third hypothesis proposes that GBM represses aberrant transcription initiation within gene bodies. Until recently, GBM could not be directly perturbed without causing genome-wide changes in DNA methylation, which limited the conclusions that could be drawn about its function. However, the Jacobsen lab has now developed tools to perform targeted DNA methylation editing in Arabidopsis. This proposal aims to use a careful series of experiments to test potential functions of GBM using these new editing tools alongside genomics assays. Using Arabidopsis as a model system, each of the three hypothesized functions of GBM will be systematically evaluated. Initial experiments will use genome-wide sequencing to identify candidate GBM genes with altered expression, splicing, or cryptic transcription in two hypomethylated lines relative to wild- type. These candidate GBM genes will then be demethylated in a wild-type background using targeted DNA methylation editing, to confirm that loss of GBM is sufficient to cause the observed phenotype. If these experiments reveal a role for GBM, potential mechanisms will also be dissected. One likely candidate for mediating GBM-dependent regulation is MBD2, which specifically binds at GBM genes in wild-type. Experiments will be performed to determine if loss of GBM disrupts MBD2 binding, and whether tethering MBD2 at artificially demethylated GBM genes can restore a normal phenotype. Other potential effectors of GBM-mediated regulation will also be explored. Taken together, these experiments will represent the most thorough investigation of the function of GBM to date. !